Unwinding of the Nascent Lagging Strand by Rep and PriA Enables the Direct Restart of Stalled Replication Forks

100

205

Collisions between cellular DNA replication machinery (replisomes) and damaged DNA or immovable protein complexes can dissociate replisomes before the completion of replication. This potentially lethal problem is resolved by cellular "replication restart" reactions that recognize the structures of prematurely abandoned replication forks and mediate replisomal reloading. In bacteria, this essential activity is orchestrated by the PriA DNA helicase, which identifies replication forks via structure-specific DNA binding and interactions with fork-associated ssDNA-binding proteins (SSBs). However, the mechanisms by which PriA binds replication fork DNA and coordinates subsequent replication restart reactions have remained unclear due to the dearth of high-resolution structural information available for the protein. Here, we describe the crystal structures of full-length PriA and PriA bound to SSB. The structures reveal a modular arrangement for PriA in which several DNA-binding domains surround its helicase core in a manner that appears to be poised for binding to branched replication fork DNA structures while simultaneously allowing complex formation with SSB. PriA interaction with SSB is shown to modulate SSB/DNA complexes in a manner that exposes a potential replication initiation site. From these observations, a model emerges to explain how PriA links recognition of diverse replication forks to replication restart.X-ray crystal structure | single-molecule FRET

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Structural mechanisms of PriA-mediated DNA replication restart

Bhattacharyya

George

Thurmes

et al. 2013

100

205

“…We chose to study BCM sensitivity in MDS42, which is deleted for all cryptic prophages, several of which contain toxic genes and recombination activities that are upregulated by BCM (15) and might affect the phenotype. Rep and PriA DNA helicases reassemble replisomes at arrested forks to restore DNA synthesis (6,21,22). MDS42 carrying rep or priA deletions were exquisitely sensitive to BCM [minimum inhibitory concentration (MIC) <15 μg/mL] (Fig.…”

Section: Resultsmentioning

confidence: 99%

Transcription termination maintains chromosome integrity

Washburn

Gottesman²

2010

110

152

DNA replication fork movement is impeded by collisions with transcription elongation complexes (TEC). We propose that a critical function of transcription termination factors is to prevent TEC from blocking DNA replication and inducing replication fork arrest, one consequence of which is DNA double-strand breaks. We show that inhibition of Rho-dependent transcription termination by bicyclomycin in Escherichia coli induced double-strand breaks. Cells deleted for Rho-cofactors nusA and nusG were hypersensitive to bicyclomycin, and had extensive chromosome fragmentation even in the absence of the drug. An RNA polymerase mutation that destabilizes TEC (rpoB*35) increased bicyclomycin resistance >40-fold. Double-strand break formation depended on DNA replication, and can be explained by replication fork collapse. Deleting recombination genes required for replication fork repair (recB and ruvC) increased sensitivity to bicyclomycin, as did loss of the replication fork reloading helicases rep and priA. We propose that Rho responds to a translocating replisome by releasing obstructing TEC.DNA polymerase/RNA polymerase collisions | pulsed-field gel electrophoresis | SOS D NA replication forks translocate 20 to 30 times faster (∼600 nt/s) than the rate of RNA polymerase (RNAP) elongation (∼20 nt/s) (1-3). This finding raises the possibility of repeated collisions between replication forks and transcription elongation complexes (TEC), even when DNA and RNA synthesis are codirectional. Collisions between replication forks and TEC can generate fork collapse and chromosomal double-strand breaks (DSBs) (4, 5). Mechanisms have evolved, therefore, to reduce collisions and to repair collapsed forks. Fork repair is promoted by multiple pathways that involve a variety of DNA repair functions (6). Recombination can restart replisomes blocked by arrested TEC (7,8). Boubakri et al. (9) have demonstrated that DNA helicases Rep, DinG, and UvrD resolve head-on collisions between TEC in rrn operons and replisomes. A recent report describes the role of transcription elongation factors DksA and GreA in preventing conflicts between replication and transcription (7, 10). In vitro, replication forks that collapse after colliding with a codirectional TEC can restart, using an R-loop (RNA-DNA hybrid) from a transcription bubble as primer (11), although it is not known if this occurs in vivo.Transcription termination isolates transcription units and prevents inappropriate expression of downstream genes. Termination in Escherichia coli is largely mediated by Rho, an RNAdependent ATPase that terminates transcription promoter-distal to unstructured and untranslated RNA (12, 13). The RNA/DNA helicase activity of Rho promotes termination by unwinding RNA/DNA hybrid in the RNAP transcription bubble (14). Inhibition of Rho with bicyclomycin (BCM) or deletion of the Rho accessory factors, NusA and NusG, massively disregulate gene expression in E. coli (15). Surprisingly, efficient transcription termination is only required to suppress expression of cryp...

“…Rep facilitates both clearance of nucleoprotein complexes ahead of forks (10,22) and PriC-directed reloading of the replication apparatus (28,29). However, the colony-forming ability of a strain lacking PriC was not reduced by RecD2 (Fig.…”

Section: Clearance Of Nucleoprotein Barriers Ahead Of Forks Protects mentioning

confidence: 99%

Protein–DNA complexes are the primary sources of replication fork pausing in Escherichia coli

Gupta

Guy

Yeeles

et al. 2013

Self Cite

105

Replication fork pausing drives genome instability, because any loss of paused replisome activity creates a requirement for reloading of the replication machinery, a potentially mutagenic process. Despite this importance, the relative contributions to fork pausing of different replicative barriers remain unknown. We show here that Deinococcus radiodurans RecD2 helicase inactivates Escherichia coli replisomes that are paused but still functional in vitro, preventing continued fork movement upon barrier removal or bypass, but does not inactivate elongating forks. Using RecD2 to probe replisome pausing in vivo, we demonstrate that most pausing events do not lead to replisome inactivation, that transcription complexes are the primary sources of this pausing, and that an accessory replicative helicase is critical for minimizing the frequency and/or duration of replisome pauses. These findings reveal the hidden potential for replisome inactivation, and hence genome instability, inside cells. They also demonstrate that efficient chromosome duplication requires mechanisms that aid resumption of replication by paused replisomes, especially those halted by protein-DNA barriers such as transcription complexes.DNA repair | genome stability | Rep | RNA polymerase | recombination